Fig: Enzyme Assay of YopH with various concentrations of enzyme (0.125, 0.5, 1, and 1.5 ng/uL), with absorbance measured at 410nm
Fig: Inhibition Assay of YopH tested with inhibitor 5852635. Absorbance measured at 410 nm. Controls with no enzyme and no inhibitor compound, and a positive control (orthovanadate) assays were performed. Standard deviation as error bars, N=2.
Top scoring ligands from CB306 library
Score
S(PLP
) S(hbond)
S(cho)
S(metal)
DE(clash)
DE(tors)
intcor
Ligand name
79.58
-39.68
3.67
0
5
0
1.65
2.08
'7676009'
78.91
-39.75
0.99
0
5.97
0
0.44
0.04
'5282931'
78.71
-42.59
2
0
4.96
0
0.36
0.06
'7724431'
76.06
-27.41
4.44
0
5.94
0
0.72
0.14
'7726180'
74.22
-26.69
4.35
0
6
0
0.79
0.06
'5175515'
73.08
-33.86
1.31
0
5.97
0
0.28
0.01
'5135469'
72.87
-39.66
2.01
0
4.84
0
0.95
0.02
'7589277'
72.75
-33.34
1.46
0
5.88
0
0.13
0
'7814264'
72.47
-43.7
0.82
0
4.66
0
0.83
0
'7761278'
72.26
-36.97
0
0
6
0
0.39
0.03
'7397377'
71.71
-15.97
3.62
0
7.97
0
1.54
0.1
'5192853'
71.42
-17.99
2.55
0
7.85
0
0.7
0.07
'7842136'
70.97
-16.08
2.85
0
7.91
0
0.56
0.01
'6433056'
70.8
-37.1
2.08
0
4.96
0
0.64
0.02
'7437091'
70.69
-38.04
1.37
0
5
0
0.77
0.06
'6407567'
70.31
-40.41
1
0
4.99
0.6
1.51
0.01
'7591231'
70.26
-48.73
1.85
0
2.97
0
0.94
0.06
'7957875'
69.44
-31.47
0.9
0
6
0
0.37
0.03
'6565654'
69.04
-37.89
1.35
0
4.99
0
1.46
0.05
'7928256'
68.92
-45.2
2.19
0
2.96
0
0.6
0.61
'5525637'
68.23
-28.85
1.35
0
5.91
0
0.07
0.02
'7907769'
67.04
-26.36
1.97
0
5.94
0
0.43
0.01
'9040855'
66.75
-26.56
1.82
0
5.98
0
0.6
0.02
'5490779'
66.5
-45.25
3.41
0
2
0
0.5
0.01
'7679150'
66.3
-31.46
0.01
0
5.99
0
0.57
0.01
'7278181'
Top scoring ligands from InHouseCompounds library
Score
S(PLP)
S(hbond)
S(cho)
S(metal)
DE(clash)
DE(tors)
intcor
Ligand name
82.59
-25.16
0.02
0
9.97
0
1.25
0.05
'5107893'
79.06
-53.29
1.1
0
3.97
0
0.93
0.2
'7796224'
75.39
-46.82
2.16
0
3.91
0.05
0.74
0.15
'7706580'
73.97
-47.78
2.54
0
3.56
0
1.77
0.35
'7752888'
Week 11 and 12
Top scoring ligands from NIHClinicalCollection library
Score
S(PLP
) S(hbond)
S(cho)
S(metal)
DE(clash)
DE(tors)
intcor
Ligand name
106.64
-71.3
4.74
0
4
0
5.92
8.14
'SAM001246803'
93
-84.87
0
0
2
0.05
4.22
4.63
'SAM001246793'
87.13
-56.51
7.39
0
2
0
1.93
0.33
'SAM001246840'
85.81
-28.1
0.06
0
9.82
0
0.71
0
'SAM001246904'
84.97
-25.06
5.51
0
7.54
0
1.83
0.94
'SAM001246552'
84.5
-47.8
0.76
0
5.99
0.05
0.87
0.11
'SAM001246721'
83.63
-35.33
1
0
7.96
0
1.49
0.52
'SAM001246684'
83.26
-42.97
3
0
5.97
0
3.48
2.4
SAM001246634'
83.04
-67.94
2.27
0
2
0
2.06
0.41
'SAM001247090'
82.49
-33.78
1
0
7.91
0
2
2.19
'SAM001246635'
82.2
-70.24
1.16
0
1.94
0
1.9
0.66
'SAM001246653'
81.17
-64.98
2
0
2
0
1.53
1.2
'SAM001247084'
80.99
-78.98
1
0
0
0
1.02
0.72
'SAM001246965'
79.38
-72.12
3.84
0
0
0
3.47
2.2
'SAM001246657'
79.37
-58.48
4
0
1.97
0
1.6
0.28
'SAM001246618'
78.9
-43.95
0.08
0
5.97
0
0.58
0.01
'SAM001246593'
78.53
-70.85
2.27
0
1
0
2.81
0.51
'SAM001247096'
78.31
-59.09
2.94
0
2
0.08
1.13
0.74
'SAM001246736'
78.3
-54.88
0.44
0
3.99
0
1.03
0.2
'SAM001246988'
77.98
-52.12
2.82
0
2.96
0
0.18
0.02
'SAM001246720'
77.77
-58.57
2.76
0
1.9
0
0.78
1.1
'SAM001246591'
77.71
-55.76
4
0
2
0
1.29
0.36
'SAM001246533'
77.61
-50.18
2.6
0
4
0
2.31
0.26
'SAM001246985'
77.6
-39.68
1.9
0
6
0
2.07
0.37
'SAM001246806'
76.1
-60.27
2.27
0
2
0.01
2.31
1.4
'SAM001246534'
75.54
-34.12
2.9
0
5.99
0
3.15
3.06
'SAM001246816'
75.21
-71.33
2.64
0
0
0
3.81
3.23
'SAM001246783'
75.11
-40.14
2.87
0
4.97
0
3.29
3.13
'SAM001246554'
74.95
-40.79
0.05
0
6
0
2.13
2.08
'SAM001246625'
74.53
-69
2.66
0
0
0
1.73
0.56
'SAM001246671'
74.02
-56.15
3.1
0
1.7
0
1.29
0.96
'SAM001246619'
73.23
-58.39
1.46
0
2
0
0.83
0.11
'SAM001246970'
72.9
-59.61
1.66
0
1.9
0.2
1.78
0.62
'SAM001246672'
72.32
-40.48
5.2
0
2.99
0
1.28
0.88
'SAM001246560'
72.16
-58.99
1
0
1.99
0
1.39
0.92
'SAM001246658'
71.95
-40.35
1.93
0.97
4.83
5.83
1.57
2.65
'SAM001246818'
71.71
-43.01
4.34
0
2.99
0
1.5
0.54
'SAM001246528'
71.63
-34.92
3
0
4.94
0
2.46
3
'SAM001246530'
71.47
-64.76
1.91
1
0
0
2.24
2.22
'SAM001246538'
71.1
-56
4.86
0
1
0
2.93
0.4
'SAM001246852'
70.74
-71.19
1
0
0
0
2.66
0.88
'SAM001246578'
70.67
-36.25
6.18
0
2.99
0
1.54
1.03
'SAM001246767'
70.45
-48.79
3.66
0
1.99
0
2.17
3.1
'SAM001246710'
70.28
-57.45
1
0
1.99
0
1.33
0.57
'SAM001247060'
69.9
-26.61
4.67
0
5
0
0.35
0.02
'SAM001247049'
Screened control ligand library:
Score
S(PLP)
S(hbond)
S(cho)
S(metal)
DE(clash)
DE(tors)
intcor
time
Ligand name
67.34
-33.19
2.71
0
4.81
85.37
0.3
83.16
26.101
'10921447pos3'
61.44
-27.22
3.81
0
3.89
192.74
1.96
196.07
25.617
'10379025pos2'
57.99
-31.28
3.03
0
2.99
0
0.89
1.42
41.363
'10880008pos1'
55.78
-30.97
2.35
0
3
0
0.19
0.14
77.403
'11151757pos7'
52.87
-25.59
3.24
0
2.99
0
0.24
0.14
73.074
'12124355pos10'
52.04
-29.86
1.82
0
2.94
0
0.52
0.13
30.427
'11031366pos4'
48.99
-52.22
0
0
0
0
1.69
0.16
48.712
'36690577neg4'
48.07
-21.85
2.76
0
3
0
0.09
0.11
10.051
'11183084pos5'
47.7
-48.29
0
0
0
0
0.34
0.08
40.811
'28082271neg3'
46.8
-47.88
0
0
0
0
0.54
0
29.708
'17399864neg2'
42.81
-20.26
1.98
0
3
0
0.74
0.11
24.961
20262261'
42.43
-30.17
1.52
0.71
1
0
0.22
0
8.522
'16654126neg1.5'
42.01
-17.76
2.21
0
3
0
0.57
0.78
13.401
'2244neg1'
40.7
-14.61
2.94
0
3
0
0.43
0.13
33.274
'10877388pos9'
37.48
-10.86
2.87
0
3
0
0
0
21.578
'13818pos6'
34.89
-9.09
2.6
0
3
0
0
0
17.615
'197063pos11'
In general most positive controls scored higher. Moved on to screening novel ligand libraries
Week 9 and 10
10/31/13
Found active site in homology model, placed dummy ligand in approximate location
Fig: Homology model made with ICM from template 1M63_A, shown as cartoon, with active site shown as sticks colored by element with green carbons, and dummy ligand glycerol shown as yellow sticks
10/29/13-10/30/13
Started Virtual Screening on target protocol
Found control ligands, ran LigPrep on them
Fig. Positive and negative control ligand CID and properties
10/26/13
Positive clone obtained from colony 8, identified after DNA sequencing and Blastn
Fig. Blastn results of aligning DNA sequencing results of colony 8 and template gene Pvivax STPP2b, 1695bp
10/22/13-10/25/13
Created Homology model for target P.vivax STPP2b using template 1M63_chainA and ICM
Fig: Homology model made with ICM from template 1M63_A, shown as cartoon
Week 7 and 8
Nice work on keeping your data updated. Try to include DNA sizes in your caption and also where exactly are you with virtual work? Thank you. -Max 10/21/13
10/18/13
A master plate was made from nine of these colonies that grew and 5ml cultures were grown up the night before and midiprepped. All nine samples were nanodropped then sent to DNA sequencing to check which were positive clones.
Figs 1, 2: Nanodrop measurements of the second and third colonies from master plate after midiprep. Measurements from all nine samples were taken, and concentrations ranged from 50 ng/ul to 110.5 ng/ul
DNA sequencing order number: 96613
10/14/13
The two plates with pNIC-Bsa4 with STPP2b in DH5-alpha had a total of eleven colonies successfully grow.
Fig. DH5-alpha with pNIC-Bsa4 + STPP2b on LB agar+kanamycin+sucrose plates
These eleven colonies should have the recombinant plasmid successfully transformed into them. A master plate was made from nine of these colonies and 5ml cultures were grown up overnight and midiprepped.
10/11/13
pNIC-Bsa4 was cut with restriction enzyme BsaI. The results were PCR cleaned up then run on a gel.
Lane 1: Empty
Lane 2: 1kb ladder
Lane 3: empty
Lane 4: pNIC-Bsa4 cut with RE BsaI
The lane with the cut plasmid has two clear bands, showing that the plasmid did cut properly at the sites it was supposed to cut at, and the SacB gene has been cut out of the plasmid, leaving the backbone and ends where our target gene STPP2b can be ligated into.
The rest of the cloning procedure was carried out, and the recombinant plasmid transformed into DH5-alpha competent cells and plated onto LB+kanamycin+sucrose plates and placed in the 37 degree incubator.
10/10/13
After pNIC-Bsa4 in DH5-alpha was grown upn LB media overnight, cells were spun down and midiprepped.
Nanodrop image of pNIC-Bsa4 plasmid concentration
Fig.: Nanodrop image of pNIC-Bsa4 plasmid concentration
A good concentration of the pNIC vector is evident from the Nanodrop.
10/09/13
Gel of PCR squared reaction
Lane2: 1kb ladder
Lane3: secondary PCR
Lane 4-7: PCR squared reaction (annealing temp. 59 degrees)
Clear bands were seen in the PCR squared reaction, and PCR cleanup was performed on the results. Moving forward to cloning. Week 5 and 6
Good data but try to add more detail into your caption and analysis. Thank you. -Max 10/07/2013
10/03/13 Gel of PCR squared reaction, changed annealing temperature to 59 degrees Lane 1: 1kb ladder Lane 2-4: PCR squared reaction
Bands are still too faint. Will compare conditions with group member and troubleshoot.
9/27/13 Gel of PCR squared reaction Lane 1: 1kb ladder Lane 2-5: PCR squared product
Very faint bands visible at proper gene length, particularly in lane 3. Redo PCR squared with annealing temperature of 59 degrees celsius that Anita used which worked.
Week 3 and 4 Ariel - ok keep moving forward with your cloning work. We want to get a clone soon. Dr. B 100113 9/20/13 Nanodrop of elute: pNIC-Bsa4 in DH5-alpha after midiprep. Negative yield, pNIC cultures have to be regrown and midiprepped
9/13/13 Lane 1: empty Lane 2: 1 kb ladder Lane 3: Secondary PCR Lane 4-7: Anita's PCR squared
9/12/13 Gel of overlap PCR Lane 1: 1kb ladder Lane 2: Secondary PCR Lane 3: Anita's secondary PCR
9/11/13 Gel of Overlap PCR for STPP2b Lane 1: 1kb ladder Lane 2: My primary PCR Lane 3: Secondary PCR (option A) Lane 4: Secondary PCR (option B) Lane 5: Anita's secondary PCR
Week 1 and 2**
Ariel - good. Also work on making some pNIC-bsa4 Dr .B 090913
Gel of Overlap PCR for STPP2b
Lane 1: empty
Lane 2: 100 bp ladder
Lane 3: primary PCR with Oligo mix
Lane 4: secondary PCR with first and last oligo
Lane 5: secondary PCR with for- and rev- tail primers
Neither secondary PCR were successful and the smear from primary PCR is very faint.
Oligonucleotides for target P. vivax STPP2b from DNAWorks:
Things To Do:
Submit to DNA Sequencing
Analyze DNA Sequence Exercise
PCR of pGBR22
Red OR Green PCR
PCR of pNIC-Bsa4 with pLIC-for and pLIC-rev primers
7/17/13
pNIC PCR gel
Fig. 5: PCR gel for pNIC plasmid with pLIC for/rev primers
Lane 1: 100bp ladder
Lane 2: Sample 1 (Lowest concentration template)
Lane 3: Sample 3 (Highest concentration template)*
Lane 4: Sample 2 (Next highest concentration template)*
Lane 5: Sample 4 (0 ng concentration template)
Lane 6: SKIP
*mistakenly loaded in non-sequential order
7/16/13
mCherry PCR gel
Fig. 4: PCR gel for pmCherry plasmid
Lane 1: SKIP
Lane 2: 100bp ladder
Lane 3: Sample 1 (0.0112 ng template with for primer VDSR1/rev primer VDSR2)
Lane 4: Sample 2 (0.112 ng template with for primer VDSR1/rev primer VDSR2)
Lane 5: Sample 3 (1.12 ng template with for primer VDSR1/rev primer VDSR2)
Lane 6: Sample 4 (0 ng template with for primer VDSR1/rev primer VDSR2)
Lane 7: Sample 5 (0.0112 ng template with M13 for/rev primer)
Lane 8: Sample 6 (0.112 ng template with M13 for/rev primer)
Lane 9: Sample 7 (1.12 ng template with M13 for/rev primer) Lane 10: Sample 8 (0 ng template with M13 for/rev primer)
Lane 6 (Sample 4) should not have a visible band present in it, and this may be evidence of contamination or some of the sample from Lane 5 moving into Lane 6
7/11/13
Failed Red PCR gel
7/9/13
pGBR22 PCR
Fig. 2: PCR gel for pGBR22 plasmid using M13 forward and reverse primers
Lane 1: SKIP
Lane 2: 100bp marker
Lane 3: Sample A (0.3 ng of template)
Lane 4: Sample B (3 ng of template)
Lane 5: Sample C (30 ng of template)
Lane 6: Sample D (0 ng of template)
7/5/13
Fig. 1 Restriction Enzyme Digest gel for pGBR22 plasmid
Enzyme Inhibition Assay
Top scoring ligands from CB306 library
Top scoring ligands from InHouseCompounds library
Week 11 and 12
Top scoring ligands from NIHClinicalCollection library
Screened control ligand library:
Week 9 and 10
10/31/13
Found active site in homology model, placed dummy ligand in approximate location
Fig: Homology model made with ICM from template 1M63_A, shown as cartoon, with active site shown as sticks colored by element with green carbons, and dummy ligand glycerol shown as yellow sticks
10/29/13-10/30/13
Started Virtual Screening on target protocol
Found control ligands, ran LigPrep on them
Fig. Positive and negative control ligand CID and properties
10/26/13
Positive clone obtained from colony 8, identified after DNA sequencing and Blastn
Fig. Blastn results of aligning DNA sequencing results of colony 8 and template gene Pvivax STPP2b, 1695bp
10/22/13-10/25/13
Created Homology model for target P.vivax STPP2b using template 1M63_chainA and ICM
Fig: Homology model made with ICM from template 1M63_A, shown as cartoon
Week 7 and 8
Nice work on keeping your data updated. Try to include DNA sizes in your caption and also where exactly are you with virtual work? Thank you. -Max 10/21/13
10/18/13
A master plate was made from nine of these colonies that grew and 5ml cultures were grown up the night before and midiprepped. All nine samples were nanodropped then sent to DNA sequencing to check which were positive clones.
Figs 1, 2: Nanodrop measurements of the second and third colonies from master plate after midiprep. Measurements from all nine samples were taken, and concentrations ranged from 50 ng/ul to 110.5 ng/ul
DNA sequencing order number: 96613
10/14/13
The two plates with pNIC-Bsa4 with STPP2b in DH5-alpha had a total of eleven colonies successfully grow.
Fig. DH5-alpha with pNIC-Bsa4 + STPP2b on LB agar+kanamycin+sucrose plates
These eleven colonies should have the recombinant plasmid successfully transformed into them. A master plate was made from nine of these colonies and 5ml cultures were grown up overnight and midiprepped.
10/11/13
pNIC-Bsa4 was cut with restriction enzyme BsaI. The results were PCR cleaned up then run on a gel.
Lane 1: Empty
Lane 2: 1kb ladder
Lane 3: empty
Lane 4: pNIC-Bsa4 cut with RE BsaI
The lane with the cut plasmid has two clear bands, showing that the plasmid did cut properly at the sites it was supposed to cut at, and the SacB gene has been cut out of the plasmid, leaving the backbone and ends where our target gene STPP2b can be ligated into.
The rest of the cloning procedure was carried out, and the recombinant plasmid transformed into DH5-alpha competent cells and plated onto LB+kanamycin+sucrose plates and placed in the 37 degree incubator.
10/10/13
After pNIC-Bsa4 in DH5-alpha was grown upn LB media overnight, cells were spun down and midiprepped.
Nanodrop image of pNIC-Bsa4 plasmid concentration
Fig.: Nanodrop image of pNIC-Bsa4 plasmid concentration
A good concentration of the pNIC vector is evident from the Nanodrop.
10/09/13
Gel of PCR squared reaction
Lane2: 1kb ladder
Lane3: secondary PCR
Lane 4-7: PCR squared reaction (annealing temp. 59 degrees)
Clear bands were seen in the PCR squared reaction, and PCR cleanup was performed on the results. Moving forward to cloning.
Week 5 and 6
- Good data but try to add more detail into your caption and analysis. Thank you. -Max 10/07/2013
10/03/13Gel of PCR squared reaction, changed annealing temperature to 59 degrees
Lane 1: 1kb ladder
Lane 2-4: PCR squared reaction
Bands are still too faint. Will compare conditions with group member and troubleshoot.
9/27/13
Gel of PCR squared reaction
Lane 1: 1kb ladder
Lane 2-5: PCR squared product
Very faint bands visible at proper gene length, particularly in lane 3. Redo PCR squared with annealing temperature of 59 degrees celsius that Anita used which worked.
Week 3 and 4
Ariel - ok keep moving forward with your cloning work. We want to get a clone soon. Dr. B 100113
9/20/13
Nanodrop of elute: pNIC-Bsa4 in DH5-alpha after midiprep.
Negative yield, pNIC cultures have to be regrown and midiprepped
9/13/13
Lane 1: empty
Lane 2: 1 kb ladder
Lane 3: Secondary PCR
Lane 4-7: Anita's PCR squared
9/12/13
Gel of overlap PCR
Lane 1: 1kb ladder
Lane 2: Secondary PCR
Lane 3: Anita's secondary PCR
9/11/13
Gel of Overlap PCR for STPP2b
Lane 1: 1kb ladder
Lane 2: My primary PCR
Lane 3: Secondary PCR (option A)
Lane 4: Secondary PCR (option B)
Lane 5: Anita's secondary PCR
Week 1 and 2**
Ariel - good. Also work on making some pNIC-bsa4 Dr .B 090913
Gel of Overlap PCR for STPP2b
Lane 1: empty
Lane 2: 100 bp ladder
Lane 3: primary PCR with Oligo mix
Lane 4: secondary PCR with first and last oligo
Lane 5: secondary PCR with for- and rev- tail primers
Neither secondary PCR were successful and the smear from primary PCR is very faint.
Oligonucleotides for target P. vivax STPP2b from DNAWorks:
Fall '13 start
Ariel - good job. - Dr. B 071713
Things To Do:
Submit to DNA Sequencing
Analyze DNA Sequence Exercise
PCR of pGBR22
Red OR Green PCR
PCR of pNIC-Bsa4 with pLIC-for and pLIC-rev primers
7/17/13
pNIC PCR gel
Lane 1: 100bp ladder
Lane 2: Sample 1 (Lowest concentration template)
Lane 3: Sample 3 (Highest concentration template)*
Lane 4: Sample 2 (Next highest concentration template)*
Lane 5: Sample 4 (0 ng concentration template)
Lane 6: SKIP
*mistakenly loaded in non-sequential order
7/16/13
mCherry PCR gel
Lane 1: SKIP
Lane 2: 100bp ladder
Lane 3: Sample 1 (0.0112 ng template with for primer VDSR1/rev primer VDSR2)
Lane 4: Sample 2 (0.112 ng template with for primer VDSR1/rev primer VDSR2)
Lane 5: Sample 3 (1.12 ng template with for primer VDSR1/rev primer VDSR2)
Lane 6: Sample 4 (0 ng template with for primer VDSR1/rev primer VDSR2)
Lane 7: Sample 5 (0.0112 ng template with M13 for/rev primer)
Lane 8: Sample 6 (0.112 ng template with M13 for/rev primer)
Lane 9: Sample 7 (1.12 ng template with M13 for/rev primer)
Lane 10: Sample 8 (0 ng template with M13 for/rev primer)
Lane 6 (Sample 4) should not have a visible band present in it, and this may be evidence of contamination or some of the sample from Lane 5 moving into Lane 6
7/11/13
Failed Red PCR gel
7/9/13
pGBR22 PCR
Lane 1: SKIP
Lane 2: 100bp marker
Lane 3: Sample A (0.3 ng of template)
Lane 4: Sample B (3 ng of template)
Lane 5: Sample C (30 ng of template)
Lane 6: Sample D (0 ng of template)
7/5/13
Lane1: SKIP
Lane2: 1kb DNA ladder
Lane3: uncut plasmid
Lane4: EcoRI Digestion Reaction
Lane5: PvuII Digestion Reaction
Lane6: EcoRI + PvuII Digestion Reaction